Cardioprotection from ischemic as well as pharmacological pre- and post- conditioning after ischemia and reperfusion (IR) injury in animal models is highly successful. Ion channels located in the mitochondria are projected as key targets for genetic and pharmacological interventions to protect the heart from IR injury. Supporting the cardioprotective role of mitochondrial ion channels, several potassium channels located at inner mitochondrial membrane have been identified and shown to be directly involved in cardioprotection from IR injury. However the molecular identity of chloride channels localized to inner mitochondrial membrane, a key anion in mitochondria, and their direct roles in mitochondrial physiology are not elucidated. Pharmacological treatment of hearts with indanyloxyacetic acid 94 (IAA-94), a known chloride intracellular channel proteins (CLICs) blocker, before IR abolished the cardioprotective effect of ischemic-preconditioning (IPC) and cyclosporine A (CsA) implicating CLIC proteins in cardioprotection. CLICs are dimorphic class of ion channel proteins with six mammalian orthologues (CLIC1-6) and a single Drosophila homolog (DmCLIC). They were identified by affinity purification using IAA-94 and channel activity of CLICs was blocked by IAA-94 and affinity purified antibodies, and promoted by low pH. We have found that IAA-94 can increase myocardial infarction in ex vivo and in vivo animal models, and also modulate mitochondrial reactive oxygen species (ROS) and capacity for calcium (CRC). Our genetic approach using CLIC null mutant flies indicated that ablation of DmCLIC protect the heart from IR injury in agreement with protective role of mammalian CLICs (CLIC4) in pulmonary hypertension. This contrasting result could arise from non-specificity of IAA-94 and/or it?s in ability to differentiate between CLIC1-6. We discovered that DmCLIC and mammalian CLIC4 and CLIC5 localizes to the cardiac mitochondria. Even though our approaches implicate CLICs in cardioprotection and mitochondrial function, the lack of direct evidence of role of mammalian CLICs, prevent them from being proposed as targets for cardioprotection. Thus, we will test the hypothesis that: 1) CLIC4 and CLIC5 proteins localize to cardiac mitoplasts; 2) where they play a direct role in cardioprotection from IR injury by getting activated due to changes in intracellular pH (pHi) during ischemia; and 3) contribute to cardioprotection by regulating mitochondrial volume, ROS, CRC and permeability transition pore (mPTP) opening. A multidisciplinary approach with cardiac specific conditional knock out mice will test our hypothesis in the following specific aims to: 1) identify molecular correlates of cardiac mitochondrial CLICs, 2) directly address the role of cardiac mitochondrial CLICs in cardioprotection, and 3) establish the role of cardiac CLICs in mitochondrial structure and function. The outcomes of this program will open the opportunity to study cardiac chloride channels at the molecular level and advance the cardiac field by: providing identity of cardiac mitochondrial chloride channel(s), defining its role in cardioprotection and possibly a mitochondrial-mediated mechanism.